UNIVERSITI PUTRA MALAYSIA

EFFECTS OF LACTOBACILLUS STRAINS AS A PROBIOTIC AND A HYPOLIPIDAEMIC AGENT FOR CHICKENS

KALAVATHY RAMASAMY

IB 2003 2

EFFECTS OF LACTOBACILLUS STRAINS AS A PROBIOTIC AND A HYPOLIPIDAEMIC AGENT FOR CHICKENS

By

KALA V ATHY RAMASAMY

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirement for the Degree of Doctor of Philosophy

August 2003

Abstract of the thesis submitted to the senate of Universiti Putra Malaysia in fulfillment of the requirement for the Degree of Doctor of Philosophy

EFFECTS OF LACTOBACILLUS STRAINS AS A PROBIOTIC AND A HYPOLIPIDAEMIC AGENT FOR CHICKENS

By

KALA V ATHY RAMASAMY

August 2003

Chairman: Professor Dr. Ho Yin Wan Institute : Bioscience

In recent years, there has been considerable interest in the beneficial effects

of probiotics (direct-fed microbials, which include Lactobacillus) to modulate the

lipid metabolism. However, the mechanism(s) involved remains unclear. A series of

experiments was carried out to investigate the ability of 1 2 Lactobacillus strains to

deconjugate bile salts and to remove cholesterol in vitro, and to assess their potential

as a pro biotic and as a hypolipidaemic agent for broilers and laying hens. Bile salt

hydrolase (BSH) activity (resulting in bile salt deconjugation) of intestinal bacteria

is closely linked to the lowering of cholesterol. The results of the in vitro studies

showed that all the 1 2 Lactobacillus strains could deconjugate sodium glychocholate

(GCA) and sodium taurocholate (TCA) bile salts, and all the strains, except L.

fermentum I 24, had a higher affinity for GCA. However, only eight strains could

deconjugate sodium taurodeoxycholate (TDCA). This indicates that the BSH of the

Lactobacillus strains is substrate specific. The 12 Lactobacillus strains showed

significant differences in their ability to reduce cholesterol from the growth medium

(27 to 85 %) with or without bile salt, indicating that bile salt is not a prerequisite for

the removal of cholesterol. Lactobacillus acidophilus I 1 6, L. crispatus I 12, L.

11

brevis C 1 7 and I 2 1 1 , and L. Jermentum I 24 and I 25 removed cholesterol from the

growth medium mainly through assimilation of cholesterol into the cells. On the

other hand, L. brevis C 1 , C 1 0, I 23 and I 2 1 8, and L. Jermentum C 1 6 removed

cholesterol through both assimilation and co-precipitation of deconjugated bile salt

with cholesterol at low pH. The Lactobacillus strains assimilated more esterified

than non-esterified cholesterol and the assimilated cholesterol was tightly bound to

the cells. Cells grown in the presence of cholesterol were more resistant to lysis by

sonication than when grown in its absence, suggesting a possible alteration of the

cell wall or membrane by the assimilated cholesterol. Cholesterol removal by the

Lactobacillus strains was also affected by Tween 80.

The feeding trials showed that the supplementation of a mixture of the 1 2

Lactobacillus cultures (LC), as a probiotic for broilers, significantly improved

growth equivalent to that provided by the antibiotic, oxytetracycline, but the feed

conversion ratio was better in LC-fed broilers. The supplementation of LC also

significantly lowered the total cholesterol, low density lipoprotein cholesterol and

triglycerides of the serum; the cholesterol of the carcass and liver; abdominal fat

deposition; and fat contents of the liver, muscle and carcass of broilers; but there

was little effect on the fatty acid compositions of the liver, muscle and carcass.

In laying hens, the supplementation of LC improved the feed efficiency and

hen-day egg production during the early stage of the laying cycle, and increased egg

weight and influenced a shift from small and medium to large and extra large eggs

throughout the laying cycle. However, LC had very little effect on improving the

fatty acid composition, and the cholesterol and total fat contents of eggs.

III

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk Ijazah Doktor Falsafah

KESAN PELBAGAI STRAIN LACTOBACILLUS SEBAGAI PROBIOTIK DAN AGEN HIPOLIPIDIMIK UNTUK A YAM

Oleh

KALA V ATHY RAMASAMY

Ogos 2003

Pengerusi : Profesor Dr. Ho Yin Wan Institut : Biosains

Sejak kebelakangan ini, kecenderungan untuk menggunakan probiotik

(mikrob makanan, termasuk Lactobacillus) dalam mengawal atur metabolisma lipid

semakin berkembang. Namun demikian, mekanisma yang terlibat masih tidak jelas.

Satu siri eksperimen telah dijalankan untuk mengkaji keupayaan 1 2 strain

Lactobacillus untuk melakukan dikonjugasi garam hempedu (garam konjugat) dan

mengurangkan kolesterol secara in vitro, serta kesannya sebagai probiotik dan agen

hypolipidimik terhadap ayam pedaging dan ayam penelur. Aktiviti enzim "bile salt

hydrolase (BSH)" (yang menyebabkan dikonjugasi garam hempedu) usus berkait

rapat dengan pengurangan kolesterol. Hasil kaj ian in vitro menunjukkan bahawa

kesemua 1 2 strain Lactobacillus berupaya melakukan dikonjugasi garam

"glychocholate" (GCA) dan garam "taurocholate" (TCA), dan kesemua strain,

kecuali L. fermentum I 24, menunjukkan afiniti yang lebih tinggi terhadap GCA.

Tetapi hanya lapan strain berupaya melakukan dikonjugasi garam

"taurodeoxycholate" . Ini menunjukkan bahawa aktiviti BSH Lactobacillus adalah

spesifik substrat. Duabelas strain Lactobacillus ini juga menunjukkan keupayaan

untuk mengurangkan kolesterol dari media kultur (25 hingga 85 %) yang ada atau

tiada garam hempedu. Pengurangan kolesterol dari media kultur oleh L. acidophilus

IV

I 1 6, L. crispatus I 12, L. brevis C 1 7 dan I 2 1 1 , dan L. fermentum I 24 dan I 25

adalah terutarnanya melalui asimilasi kol estero 1 oleh sel. Pengurangan kolesterol

oleh L. brevis C 1 , C 1 0, I 23 dan I 2 1 8, dan L. fermentum C 1 6 pula, adalah melalui

asimilasi dan juga ko-mendakan gararn hempedu tak berkonjugat bersarna kolesterol

pada pH yang rendah. Strain Lactobacillus mengasimilasi lebih banyak kolesterol

ester berbanding dengan kolesterol bebas dan kolesterol yang diasimilasi didapati

terikat dengan kuat pada sel. Sel yang ditumbuhkan bersarna kolesterol juga lebih

resistan kepada sonikasi, mencadangkan bahawa pengubahsuaian pada dinding atau

membran sel berlaku setelah mengasimilasi kolesterol. Pengurangan kolesterol oleh

strain Lactobacillus juga bergantung pada Tween 80.

Hasil kaj ian in vivo menunjukkan bahawa carnpuran 12 strain Lactobacillus

(LC), sebagai probiotik pada ayarn pedaging dapat meningkatkan berat badan sarna

seperti antibiotik "oxytetracycline", tetapi kadar penukaran makanan ayarn adalah

lebih baik pada ayarn yang di beri LC. Penarnbahan LC pada ayarn juga dapat

menurunkan paras "total" kolesterol, " low density lipoprotein" kolesterol dan

trigliserida di serum; kandungan kolesterol pada karkas dan hati; lemak berlebihan

pada bahagian abdomen; dan kandungan lemak pada hati, otot dan karkas; tetapi

tidak berupaya mengubah profil asid lemak pada hati, otot dan karkas.

Ayarn penelur yang di beri LC dapat meningkatkan kadar penukaran

makanan dan produksi telur pada peringkat awal peneluran serta dapat menghasilkan

telur yang lebih berat dan saiz yang lebih besar sepanjang proses peneluran. Narnun

demikian, LC kurang berkesan untuk mengubah profil asid lemak, atau menurunkan

paras kolesterol dan lemak di telur.

v

ACKNOWLEDGEMENTS

I wish to express my deep appreciation and most sincere gratitude to the

chairman of the supervisory committee, Professor Dr. Ho Yin Wan, for her

invaluable guidance and advice, endless support, patience, and encouragement

throughout the duration of this study and for her critical analysis, constructive

criticism and helpful suggestions during the preparation of my thesis.

I am deeply grateful and indebted to Associate Professor Dr. Norhani

Abdullah and Dr. Clemente Michael Wong, who are members of the supervisory

committee, for their kind assistance, advice and guidance throughout the course of

my work and in the preparation of the thesis.

Special appreciation goes to Tan Sri Dato Dr. Syed lalaludin Syed Salim

(who was a member of the supervisory committee till his retirement in 200 1 ) for his

wise counsel, support and constant encouragement.

My heartfelt appreciations are extended to Madam Haw Ah Kam, Mr.

Khairul Kamar Bakri, Mr. Nagayah Muniandy, Mr. livanathan Arumugam and Mr.

Paimon Lugiman, staff of the Enzyme and Microbial Technology Laboratory, and

Mr. Saparin Denim and Mr. Ibrahim Mohsin, staff of Animal Nutrition Laboratory,

for their technical support and kind assistance. Thanks are also due to Dr. Goh Y ong

Meng for his assistance on the preparation of samples for the fatty acid

determination using Gas Chromatography.

VI

I wish to extend my sincere thanks to my post graduate friends Chin Chin,

Latiffah, Lan, Darlis, Vicky, Wan, Thongsuk, Lee, Pit Kang and Sidieg for their

friendship, support, encouragement and their sense of humor that made the many

hours in the laboratory very pleasant, which contributed to the successful completion

of this work.

Finally, very special thanks are due to my family for their unconditional

love, untiring patience, support and encouragement, which inspired and motivated

me throughout the course of this study.

Vll

I certify that an Examination Committee met on 6th August 2003 to conduct the final examination of Kalavathy Ramasamy on her Doctor of Phliosophy thesis entitled "Effects of Lactobacillus Strains as a Probiotic and a Hypolipidaemic Agent for Chickens" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulation 1 98 1 . The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

ABDUL RAZAK ALlMON, Ph.D. Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman)

HO YIN WAN, Ph.D. Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Member)

NORHANI ABDULLAH, Ph.D. Associate Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Member)

CLEMENTE MICHAEL WONG, Ph.D. Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

HYUNG TAl SHIN, Ph.D. Professor Department of Food and Bioresources Faculty of Life Science and Technology Sung Kyun Kwan University 300 Chunchun-Dong, Jangan-Ku Suwon 440-746, Republic of Korea (Independent Examiner)

ProfessorlDeputy an School of Graduate Studies Universiti Putra Malaysia

Date: .- 4 SEP 2003

Vlll

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee are as follows:

HO YIN WAN, Ph.D. Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Chairman)

NORHANI ABDULLAH, Ph.D. Associate Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Member)

CLEMENTE MICHAEL WONG, Ph.D. Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

ix

AINI IDERIS, Ph.D. ProfessorlDean School of Graduate Studies Universiti Putra Malaysia

Date: f! 6 SEP 2003

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations

and citations which have been duly acknowledged. I also declare that it has not been

previously or concurrently submitted for any other degree at UPM or other

institutions.

x

KALA V ATHY RAMASAMY

Date: 2.:1./4/ 0 3

TABLE OF CONTENTS

Page

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 ABSTRAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... IV ACKNOWLEDGEMENTS . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI APPROVAL . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . V11l DECLARATION . . . . . . ........................................................................ x LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. , XIV LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XV11 LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. xx

CHAPTER

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2 . 1 Poultry Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5 2.2 Global Challenges in the Modem Poultry Industry . . . . . . . . . .. , 6 2.3 Lipids and Human Health 8 2 .4 Lipids in Broiler Meat and Eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0

2 .4. 1 Lipid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 10 2.4.2 Fat deposition, Cholesterol and Fatty Acids . . . . . . . . . . . . 1 2 2.4.3 Strategies in Improving the Lipid Content i n Broiler

Meat . . . . . . . . . . . . ... .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.4.4 Strategies in Improving the Lipid Content in

Eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1 6 2.4.5 Biological Methods in Improving Lipids in Broiler

Meat and Eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1 7

2.5 Antibiotics in Poultry Production : Benefits and Risks . . . . . . 1 8 2.6 Probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2.6. 1 Contributions of the Intestinal Microflora . . . . . . . . . . . . . .. 22 2.6.2 Definition .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 2.6.3 Probiotics Currently in Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 2.6.4 Mode of Action of Probiotics .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2.6.5 Selection Criteria for Probiotics .. . . . . . . . . . . . . . . . . . . . . . . . . . 28 2.6.6 Benefits of Probiotics on Poultry Performance . . . . . . . . . 28

2.7 Hypocholesterolaemic Effect of Lactic Acid Bacteria . . . . . . .. 33 2.8 Bile Salt Deconjugation of Lactic Acid Bacteria . . . . . . . . . . . . .. , 36

2.8 . 1 Enterohepatic Circulation of Bile Acids . . . . . . . . . . . . . . . . . 36 2 .8 .2 Significance of Bile Salt Deconjugation by the Lactic

Acid Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 37

3 BILE SALT HYDROLASE ACTIVITY OF LACTOBACILLUS CULTURES FROM CHICKEN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 3 . 1 Introduction . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 3.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1

Xl

3.2. 1 Source and Maintenance of Lactobacillus Strains . . . . . . 4 1 3 .2.2 Bile Salt Deconjugation by Lactobacillus Strains 42 3 .2.3 Kinetics of Bile Salt Deconjugation . . . . . . . . . . . . . . . . . . . . . . 45 3 .2.4 Bile Tolerance Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

3 .3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 3.3 . 1 Morphological Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 3 .3 .2 Bile Salt Deconjugation by Lactobacillus Strains . . . . . . 47 3.3 .3 Kinetic Parameters of Bile Salt Deconjugation . . . . . . . . . 6 1 3 .3 .4 Bile Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1

3 .4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

4 CHOLESTEROL-REDUCING ABILITY OF LACTOBACILLUS STRAINS IN VITRO AND THE MECHANISM (S) INVOL VED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 4. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 4.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

4.2. 1 Preliminary Study of Cholesterol Reduction by 1 2 Lactobacillus Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

4.2.2 Effects of Various Bile Salt Concentrations on the Reduction of Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1

4.2.3 Effects of Various Concentrations of Tween 80 on the Reduction of Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

4.2.4 Effect of Cholesterol on Growth of Lactobacillus Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

4.2.5 Quantitative Analysis of Cholesterol in the Culture Supernatant and Bacterial Cell Pellet of Three Lactobacillus Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

4.2.6 Qualitative Analysis on the Assimilation of Cholesterol by Lactobacillus Strains . . . . . . . . . . . . . . . . . . . . . 85

4.2.7 Effects of Cholesterol and Bile Salts on Lysis of Lactobacillus by Sonication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

4.2.8 Effects of pH and Bile Salts on Solubility of Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 4.3 . 1 Reduction of Cholesterol in Growth Media by 1 2

Lactobacillus Strains . . . . . . . . . .. . . . . . .. .. . .. . . . ... . . . . . . . . . . . 88 4.3.2 Effects of Bile Salt on Cholesterol Reduction . . . . . . . . . . 90 4.3.3 Effects of Concentrations of Tween 80 on Cholesterol

Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1 4.3 .4 Effect of Cholesterol on Growth of Lactobacillus

Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 4.3.5 Quantitative Analysis of Cholesterol in the Culture

Supernatant and Cell Pellet of Lactobacillus Strains . . . 96 4.3.6 Qualitative Analysis of Cholesterol in Cell Pellets of

Lactobacillus Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 4.3.7 Effect of Cholesterol and Bile Salts on Lysis of

Lactobacillus by Sonication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 04 4.3.8 Influence of pH and Bile Salts on Solubility of

Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 07

xii

5 EFFECTS OF LACTOBACILLUS CULTURES ON BROILER CHICKENS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 7 5 . 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 7 5.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 8

5.2. 1 Animals and Rearing Management . . . . . . . . . . . . . . . . . . . . . . . 1 1 9 5.2.3 Experiment II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 22 5.2.4 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 29

5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 29 5 .3 . 1 Experiment I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 5 .3 .2 Experiment II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 32

5.4 Discussion ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

6 EFFECTS OF LACTOBACILLUS CULTURES ON LAYING HENS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1 57 6. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 57 6.2 Materials and Methods .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 58

6.2. 1 Animals and Rearing Management . . . . . . . . . . . . . . . . . . . . . .. 1 58 6.2.2 Dietary Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 59 6.2.3 Layer Performance and Production . . . . . . . . . . . . . . . . . . . . . . . 1 59 6.2.4 Egg Quality and Egg Storage Test . . . . . . . . . . . . . . . . . . . . . . .. 1 6 1 6.2.5 Yolk Total Lipids, Fatty Acid Composition and

Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 1 63 6.2.6 Statistical Analysis . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. 164

6.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 1 64 6.3 . 1 Ambient Temperature and Relative Humidity . . . . . . . . . . 1 64 6.3.2 Layer Performance and Production . . . . . . . . . . . . . . . . . . . . . . . 1 65 6.3.3 Egg Quality and Storage Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 75 6.3.4 Egg Yolk Cholesterol, Total Lipids and Fatty Acid

Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 80 6.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1 85

7 GENERAL DISCUSSION AND CONCLUSIONS . . . . . . . . . . . . . . ... 194 7. 1 General Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 7.2 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 208 VITA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 234

Xlll

LIST OF TABLES

Table

1 Possible modes of actions of probiotics

2 Criteria for an effective pro biotic strain

3 Hypocholesterolaemic effects of lactic acid bacteria on various

Page

27

29

hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

4 Lactobacillus strains (from chicken) used in the study . . . . . . . . . . . . . . . . . 42

5 Bile salt hydrolase (BSH) activity of Lactobacillus strains on MRS + sodium taurodeoxycholate (MRS + TDCA) agar plates . . . . . . . . . . . . . 5 1

6 Comparison of the deconjugation of sodium taurocholate and sodium glychocholate by 1 2 Lactobacillus strains . . . . . . . . . . . . . . . . . . . . . . 59

7 Kinetics of bile salt deconjugation by L. brevis C 1 0 from 2 to 24 h of incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 63

8 Kinetics of bile salt deconjugation by L. fermentum C 16 from 2 to 24 h of incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 65

9 Kinetics of bile salt deconjugation by L. acidophilus I 26 from 2 to 24 h of incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 67

1 0 Kinetics of bile salt deconjugation by L. acidophilus I 1 6 from 2 to 24 h of incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 69

1 1 Growth of Lactobacillus strains in MRS broth and MRS with 0.3 % bile salt at 4 h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 7 1

1 2 Reduction of cholesterol in growth media by 1 2 Lactobacillus strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

1 3 Effects of bile salt concentrations on cholesterol reduction by Lactobacillus strains . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1

1 4 Effects of Tween 80 concentrations on cholesterol reduction by Lactobacillus strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 92

1 5 Comparison of growth of three Lactobacillus strains in various growth media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 94

1 6 Percentages of cholesterol reduced in the MRSC and MRSBC supernatants and percentages of cholesterol assimilated in the cell 97 pellets of three Lactobacillus strains . . ........ . . . . .... . . . . ...... . . . . . .... .

xiv

17 Fluorescence intensity of cell pellets of Lactobacillus strains grown in various media and stained with filipin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

1 8 Fluorescence intensity of cell pellets of Lactobacillus strains grown in various media and stained with Nile Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 1

19 Effects of cholesterol and bile salts on lysis of Lactobacillus by sonication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 05

20 Composition of the basal diets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 1 2 1

2 1 Effects of Lactobacillus cultures (LC) or oxytetracycline on body weight, weight gain, feed intake and feed to gain ratio of broiler chickens for 42 days . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 1

22 Effects of Lactobacillus cultures (LC) on body weight, weight gain and feed to gain ratio of broiler chickens for 42 days . . . . . . . . . . . . . . . . . . 1 33

23 Percentage by weight of organs from broiler chickens fed diets with or without Lactobacillus cultures (LC) from 2 1 to 42 days of age . . . 1 34

24 Abdominal fat deposition of broiler chickens fed with or without Lactobacillus cultures (LC) from 2 1 to 42 days of age . . . . . . . . . . . . . . . .. 135

25 Serum lipid concentrations in broiler chickens fed with or without Lactobacillus cultures (LC) from 2 1 to 42 days of age . . . . . . . . . . . . . . . 140

26 Effects of Lactobacillus cultures (LC) on cholesterol contents of carcass, liver and muscle of broiler chickens at 42 days of age . . . . . . . 14 1

27 Effects of Lactobacillus cultures (LC) on fat contents of carcass, liver and muscle of broiler chickens at 42 days of age . . . . . . . . . . . . . . . . . 14 1

28 Fatty acid composition of carcass from broilers supplemented with or without Lactobacillus cultures (LC) at 42 days of age . . . . . . . . . . . . .. 143

29 Fatty acid composition of liver from broilers supplemented with or without Lactobacillus cultures (LC) at 42 days of age . . . . . . . . . . . . . . . . . 144

30 Fatty acid composition of muscle from broilers supplemented with or without Lactobacillus cultures (LC) at 42 days of age . . . . . . . . . . . . .. 1 45

3 1 Composition of the basal diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 60

32 Egg size distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1 6 1

33 Effects of Lactobacillus cultures (LC) on feed intake, feed efficiency, hen-day egg production and mortality of laying hens from 20 to 68 weeks of age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 66

xv

34 Effects of Lactobacillus cultures (LC) on egg weight and egg mass of laying hens from 20 to 68 weeks of age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 70

35 Effects of Lactobacillus cultures (LC) on egg size of laying hens from 20 to 68 weeks of age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 73

36 Effects of Lactobacillus cultures (LC) on egg quality of hens from 20 to 35 weeks of age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 76

37 Cholesterol contents of eggs from hens supplemented with or without Lactobacillus cultures (LC) at 24, 28, 32 and 68 weeks of age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8 1

38 Total lipid contents of eggs from hens supplemented with or without Lactobacillus cultures (LC) at 24, 28 and 32 weeks of age . . . . . . . . . . . . 1 8 1

39 Fatty acid composition of �ggs from hens supplemented with or without Lactobacillus cultures (LC) at 24 weeks of age . . . . . . . . . . . . . . . 1 82

40 Fatty acid composition of eggs from hens supplemented with or without Lactobacillus cultures (LC) at 28 weeks of age . . . . . . . . . . . . . . . 1 83

4 1 Fatty acid composition of eggs from hens supplemented with or without Lactobacillus cultures (LC) at 32 weeks of age . . . . . . . . . . . . . . . 1 84

xvi

Figure 1

2

3

4

5

6

7

8

9

10

11

12

13

LIST OF FIGURES

Cell morphology of Lactobacillus strains observed using light microscopy . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Colonies of Lactobacillus strains on MRS agar ............................ ..

Plate assay showing high bile salt hydrolase (BSH) activity of L. Jermentum C 16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Plate assay showing high bile salt hydrolase (BSH) activity of L. brevis C 1 .................................................................. .

Plate assay showing high bile salt hydrolase (BSH) activity of L. brevis C 10 ................................................... ' " . . . . . . . . . . .

Plate assay showing no bile salt hydrolase (BSH) activity of L. brevis C 17 .................................................................. .

Plate assay showing low bile salt hydrolase (BSH) activity of L. crispatus I 12 and L. brevis I 23 .......................................... .

Plate assay showing absence of bile salt hydrolase (BSH) activity in L. Jermentum I 24 and L. acidophilus I 26 ............................ .

Precipitates due to bile salt hydrolase (BSH) activity as observed under the light microscope . . . . . . . . . . . . " . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Deconjugation of sodium glychocholate (OCA) and sodium taurocholate (TCA) by Lactobacillus strains . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Orowth and changes in pH, and dissappearance of conjugated bile salt in MRS broth supplemented with sodium taurocholate (TCA) and sodium glycocholate (GCA) of L. brevis C 10 from 0 to 24 h of incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Orowth and changes in pH, and dissappearance of conjugated bile salt in MRS broth supplemented with sodium taurocholate (TCA) and sodium glycocholate (OCA) of L. Jermentum C 16 from 0 to 24 h of incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Orowth and changes in pH, and dissappearance of conjugated bile salt in MRS broth supplemented with sodium taurocholate (TCA) and sodium glycocholate (OCA) of L. acidophilus I 26 from 0 to 24 h of incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . .

xvii

Page

48

49

52

53

54

55

56

57

58

60

64

66

68

1 4 Growth and changes in pH, and dissappearance o f conjugated bile salt in MRS broth supplemented with sodium taurocholate (TCA) and sodium glycocholate (GCA) of L. acidophilus I 16 from 0 to 24 h of incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 70

1 5 Growth of L. brevis C 1 0, L. acidophilus I 26 and L. acidophilus I 1 6 in four different media with or without cholesterol 95

16 Fluorescence micrographs of cell pellets of L. acidophilus I 26 stained with filipin . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 99

1 7 Fluorescence micrographs o f cell pellets of L . acidophilus I 26 stained with Nile Red . . . . .. . . . . . . . . . . . . .... .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 1 02

1 8 Fluorescence micrographs of cell pellets of L. brevis C 1 0 stained with Nile Red . . . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . ... . . . . .. 1 03

1 9 Influence of pH and bile salts on solubility of cholesterol . . . . . . . . . . . . . 1 06

20 Abdominal fat depositions of broiler chickens at 42 days of age fed without or with Lactobacillus cultures . . . ................................ 1 36

2 1 Fat depositions at different areas in broiler chickens at 42 days of age fed without or with Lactobacillus cultures . . . . . . . . . . . . . . .. . . . . . . . . . . 1 37

22 Fat deposited on the skin of broiler chickens at 42 days of age fed 1 3 8 without or with Lactobacillus cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

23 Effect of Lactobacillus cultures (LC) on feed efficiency of laying hens from 20 to 68 weeks of age .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 67

24 Effect of Lactobacillus cultures (LC) on egg production of laying hens from 20 to 68 weeks of age . . . . ... . . . . . ... . . . ... . . . . . . . . . . . . . . . . . . . . . 1 68

25 Effect of Lactobacillus cultures (LC) on egg weight of laying hens from 20 to 68 weeks of age . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . .. . . . . .... . . 1 7 1

26 Effect of Lactobacillus cultures (LC) on egg mass of laying hens from 20 to 68 weeks of age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . 1 72

27 Effect of Lactobacillus cultures (LC) on egg size of laying hens from 20 to 44 weeks of age . . . . . . . . . . . . . . ... . .. . . . . . . . . . . . . . . . . . . . . . . . . . . ... 1 74

28 Effect of Lactobacillus cultures (LC) on egg size of laying hens from 45 to 68 weeks of age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 74

29 Effect of Lactobacillus cultures (LC) and storage time on internal 177 egg quality of hens from 20 to 68 weeks of age . . . . . . . . . . . . . . . . . . . . . . . . .

xviii

30 Internal egg quality of a fresh egg and an egg that was stored for 7 days from a top view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1 78

3 1 Internal egg quality of a fresh egg (A) and an egg that was stored for 7 days (B) from a lateral view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 79

XIX

AAP ADP AFTA AOAC ATP BSH CFU cm CP d FAME FAO FDA g GC GCA GRAS h H202 HACCP HBA HDL HMG CoA HPLC HU IDL IU kg KIC KOH I LABIP LC LDL M m mg min MJ mRNA MRS MRSB MRSC MRSBC MRS-TDCA MUFA NaCl NaOH

LIST OF ABBREVIATIONS

Aminoantipyrine Adenosine diphosphate Asean Free Trade Centre Association of Official Analytical Chemists Adenosine triphosphate Bile salt hydrolase Colony forming unit centimetre Cell pellet Day Fatty acid methyl ester Food and Agriculture Organisation Food and Drug Administrations gram Gas Chromatography Sodium glychocholate Generally Recognized as Safe hour Hydrogen peroxide Hazzard Analysis Critical Control Points Hydroxybenzoic acid High density lipoprotein Hydroxymethylglutaryl coenzyme A High Performance Liquid Chromatography Haugh unit Intermediate density lipoprotein International Unit kilogram a-ketoisocaproic acid Potassium hydroxide litre International Platform for Lactic Acid Bacteria A mixture of 12 Lactobacillus cultures Low density lipoprotein Molar metre milligram minute megajoules Messenger Ribonucleic Acid Man Rogoso Sharpe MRS containing bile salt MRS containing cholesterol MRS containing bile salt and cholesterol MRS agar supplemented with 0.5 % sodium taurodeoxycholate Monounsaturated fatty acids Sodium chloride Sodium hydroxide

xx

ND OD OTe PTA PPLO PUFA SAS SCFA SFA ST TCA TDCA tRNA UFA �g �l VLDL W WHO

No data Optical density Oxytetracycline Phototungstic Acid Pleuropneumonia-like organism Polyunsaturated fatty acids Stastical Analysis Software Short chain fatty acids Saturated fatty acids Supernatant Sodium taurocholate Sodium taurodeoxycholate Transfer Ribonucleic Acid Unsaturated fatty acids microgram micro litre Very low density lipoprotein Watt World Health Organisation

XXi

CHAPTER!

INTRODUCTION

The worldwide poultry industry provides a substantial proportion of the

nutritional requirement of the human population. Poultry meat is perceived to be

lean and low in cholesterol, so it may come as a surprise to learn that poultry

scientists and producers are increasingly concerned about the amount of fat present

in chicken meat. Chambers et al. (1981), Lin (1981) and Havenstein et al. (1994)

reported that, as a result of selection strategy for body weight gain or growth rate,

modem fast-growing broilers have been found to contain about four times higher

amounts of abdominal fat than those in the 1960s. Eggs have also been viewed with

suspicion today because of their high cholesterol content (Stadelman, 1999). In the

US, egg consumption has declined from 256 eggs per capita per year in 1985 to 235

in 1995 (USDA, 1997). The lipid composition of animal products is a primary

consumer concern as high fat and cholesterol intakes have been implicated to

contribute to coronary heart disease, the most common chronic illness in developed

countries. To the poultry producers, on the other hand, excess fat is an economic

burden, as fat is lost during processing of the carcass or of the meat, resulting in

lower meat yields and, furthermore, the discarded abdominal fat and visceral fat

increases waste management problems. This has put the poultry production system

under pressure and, therefore, much attention is now directed towards producing

healthier meat and eggs such that the lipid fraction is improved (reduced cholesterol

and fat and improvement of the fatty acid make-up). Animal feed strategies, genetic

selections, and gene manipulation are some of the techniques that have been

developed to alter the lipid composition in broilers (Jimenez-Colmenero, 2000) and

egg yolk (Hargis, 1988). However, very often these techniques are cost prohibitive

or may impair performances and, therefore, not economically feasible to be applied

at commercial scale. Animal welfare and environmental issues may also be linked in

the application of these techniques.

Performance and economic returns are one of the main concerns of the

commercial poultry industry. To achieve these goals, very often, intensive farming

systems are adopted, subjecting broilers and laying hens to various stressful

situations. Stress may lower the body's defense mechanism and create an imbalance

in the intestinal microflora (Fuller, 1 999), which in tum increases susceptibility to

infectious diseases, resulting in poor performance. Efforts to prevent or reduce avian

diseases include improved management practices, but inevitably at a cost, because

this requires high quality feed manufacturing and feeding systems where the

environment and the feed are relatively pathogen-free (Zhang-Barber et ai., 1 999).

The benefits of incorporating antibiotic growth promoters in animal feeds are well

substantiated (Bedford, 2000). These products have been used for many years by

the poultry industry and have proved to be an effective way of enhancing animal

status, uniformity and production efficiency. The Union of Concerned Scientists

recently estimated that, each year, 1 1 .2 million kg of antimicrobials are given to

animals for non-therapeutic purposes, and 900,000 kg are given for theraphy, thus, it

is fair to state that substantial amounts of antimicrobials are administered to food

animals for growth promotion and feed efficiency in the absence of known disease

(Gorbach, 2001 ). However, the use of antibiotics as growth promoters is severely

restricted or totally banned in poultry production in many countries, largely because

of concern on the development of resistant bacterial strains and residual toxicity in

2